Research ArticleLong-Lasting Anti-Inflammatory Activity of HumanMicrofragmented Adipose Tissue

Sara Nava,1 Valeria Sordi ,2 Luisa Pascucci,3 Carlo Tremolada ,4,5 Emilio Ciusani,6

Offer Zeira,7 Moris Cadei,8 Gianni Soldati ,9 Augusto Pessina ,10 Eugenio Parati,1

Mark Slevin ,5,11,12 and Giulio Alessandri 1

1Cellular Neurobiology Laboratory, Department of Cerebrovascular Diseases, Fondazione IRCCS Neurological Institute C. Besta,Milan, Italy2Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy3Department of Veterinary Medicine, University of Perugia, Perugia, Italy4Image Institute, Milan, Italy5School of Healthcare Science, John Dalton Building, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK6Laboratory of Clinical Pathology and Neurogenetic Medicine, Fondazione IRCCS Neurological Institute C. Besta, Milan, Italy7San Michele Veterinary Hospital, Tavezzano con Villavesco, Lodi, Italy8Section of Pathological Anatomy DMMT, University of Brescia, Brescia, Italy9Swiss Stem Cell Foundation, In Pasquée 23, Gentilino, CH-6925 Lugano, Switzerland10Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy11Weifang Medical University, Weifang, China12University of Medicine and Pharmacy, Targu Mures, Romania

Correspondence should be addressed to Giulio Alessandri; giulio.alessandri@istituto-besta.it

Received 7 August 2018; Accepted 4 December 2018; Published 19 February 2019

Academic Editor: Marcin Majka

Copyright © 2019 Sara Nava et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Over the last few years, human microfragmented adipose tissue (MFAT), containing significant levels of mesenchymal stromalcells (MSCs) and obtained from fat lipoaspirate (LP) through a minimal manipulation in a closed system device, has beensuccessfully used in aesthetic medicine as well as in orthopedic and general surgery. Interestingly, in orthopedic diseases,this ready-to-use adipose tissue cell derivative seems to have a prolonged time efficacy even upon a single shot injectioninto osteoarthritic tissues. Here, we investigated the long-term survival and content of MSCs as well the anti-inflammatoryactivity of LP and its derived MFAT in vitro, with the aim to better understand a possible in vivo mechanism of action.MFAT and LP specimens from 17 human donors were investigated side by side. During a long-term culture in serum-freemedium, we found that the total cell number as well the MSC content in MFAT decreased more slowly if compared tothose from LP specimens. The analysis of cytokines and growth factors secreted into the conditioned medium (CM) wassimilar in MFAT and LP during the first week of culture, but the total amount of cytokines secreted by LP decreasedmuch more rapidly than those produced by MFAT during prolonged culture (up to 28 days). Similarly, the addition ofMFAT-CM recovered at early (3-7 days) and late stage (14-28 days) of culture strongly inhibited inflammatory function ofU937 monocyte cell line, whereas the anti-inflammatory activity of LP-CM was drastically reduced after only 7 days ofculture. We conclude that MFAT is an effective preparation with a long-lasting anti-inflammatory activity probablymediated by a long-term survival of their MSC content that releases a combination of cytokines that affect severalmechanisms involved in inflammation processes.

HindawiStem Cells InternationalVolume 2019, Article ID 5901479, 13 pageshttps://doi.org/10.1155/2019/5901479

1. Introduction

Autologous use of adipose mesenchymal stem/stromal cells(MSCs), or the stromal vascular fraction (SVF) isolatedfrom liposuction of fat tissue, has slowly gained supportfor the treatment of a variety of pathological conditionsfrom osteoarthritis through skin wound healing to strokeand brain injury [1]. With very few or none apparent sideeffects and a potential tissue regenerative capacity, thesefat-derived “bioreactors” could hold the key to next-generation therapies being more effective in recreation oflike-for-like three-dimensional tissue repair. SVF can act as athree-dimensionalmatrix or scaffold containing activated cel-lular components including adipocytes, pericytes/pericyte--derived MSCs, and potentially “angiogenic” endothelial cells(ECs) [2, 3]. To date, a detailed understanding of the mecha-nisms through which these biological materials are able tomoderate tissue repair is required to work hand in hand withan appreciation of the safety of such therapies.

In particular, the adipose MSC component of these SVFshas been highlighted in most detail, undergoing consider-ation for treatment of osteoarthritis and cartilage repair [4,5], anti-inflammatory stroke therapy, and treatment for Par-kinson’s disease [6, 7]. In addition, it has shown promise forthe treatment of musculoskeletal regeneration [8] and treat-ment of complex anal fistula [9].

The anti-inflammatory and cell protective properties ofthe fat tissue are of great interest, in particular the MSC secre-tome which contains specific anti-inflammatory and immu-nosuppressive cytokines and growth factors includingiNOS, IDO, PGE2, TSG6, HO1, TGF-β, and galectins [10–12], but also contains extracellular vesicles (EVs) whichrecapitulate some MSC functions. Importantly, MSC-derived EVs have been shown to retain regenerative andanti-inflammatory properties and thus proposed to be usedas cell-free therapies [13, 14]. The specific microenvironmentwithin inflammatory tissue dictates MSC response and ulti-mately phenotypical variations; therefore, it is critical tounderstand MSC homing and secretion in order to postulatepossible therapeutic applications [10, 15].

While several mechanical and enzymatic protocols havebeen used to prepare fat MSCs or the most impure SVF,involving centrifugation, washing, and filtration [2], mostrecently, Tremolada and colleagues [16] developed a relativelysimple self-containedmechanical technique to create amicro-fragmented adipose-derived fraction (MFAT) through anenzyme-free technology, able to convert lipoaspirate (LP) intoMFAT using a device named Lipogems®. This techniquereduces the size of the adipose tissue clusters bymeans ofmildmechanical forces and eliminates oil and blood residue. Thetechnique is gentle and provides microfragmented fat in ashort time (15-20min), without expansion and/or enzymatictreatment. Through this technology, it was demonstrated thatMFAT contains a significant number of MSCs that can bedirectly injected into patients [17]. This nonexpanded MFAThas been shown to possess regenerative properties, particu-larly when injected into inflammatory or ischemic tissues[16, 18]; recently, it has been successfully used in aestheticmedicine as well as in orthopedic diseases. Interestingly, inorthopedic diseases, this ready-to-use adipose tissue cell

derivative has shown a very prolonged time efficacy even upona single shot injection into dogs with osteoarthritic disease[18]. Our group has shown that this biomaterial could blockthe proinflammatory activities of U937 macrophages/mono-cyte cell line by reducing their ability to bind activate ECs[19] while intraperitoneal injection of MFAT significantlyattenuated inflammation following caecal ligation in a mousemodel of sepsis [18]. Based on these experimental and clinicalresults, in this work, we aimed to identify the mechanisticdetail differentiating MFAT from the standard LP. Morespecifically, we investigated the long-term survival and con-tent of MSCs as well the anti-inflammatory activity of LPand its derived MFAT. We also analyzed their secretomein vitro. We found that MFAT specimens, cultured underserum-free conditions, contained a significant amount ofMSCs and have an impressive capacity to secrete moleculeswith anti-inflammatory properties whose activity lasts forweeks; vice versa, MSC content and secretome activity of LPcounterpart, under the same culture conditions, decay rapidly(within a week).

2. Materials and Methods

2.1. MFAT Processing from Lipoaspirate. According to thepolicies approved by the Institutional Review Boards forHuman Studies local ethical committees (IRB 48/2013, Isti-tuto Neurologico Carlo Besta), 17 different fat donors wereinvestigated. In this study, LPs were obtained from patientsundergoing plastic surgery; written informed consent wasobtained from all donors. MFAT specimens were preparedfrom LP counterpart of the same donor, as previouslydescribed [17]. Briefly, 100ml of LP was obtained from eachpatient, and 50ml of LP was used for MFAT preparation byusing a standard 225ml Lipogems® device (provided byLipogems® International, Milan, Italy). The LP collected bysyringe is pushed into Lipogems® device through a filter fora first cluster reduction; afterwards, the five stainless steelmarbles inside the device are shaken to disaggregate fat mate-rial producing cell clusters and microfragmented fat tissuethat migrated to the top of device, while blood contaminatingcells and undesired fat residues are removed by a gravitycounterflow of saline solution. When the solution inside thedevice appears yellow and clear, the device was turned upsidedown and a second microfragmentation of the tissue wasobtained by pushing the adipose clusters with a syringethrough a size reduction filter. At the end of this procedure,MFAT product was aspirated by a syringe connected withthe device and was ready for investigation.

2.2. Preparation of Conditioned Medium (CM) from MFAT(MFAT-CM), LP (LP-CM), and Their Isolated MSCs(MSCs-CM). Specimens of LP and its counterpart MFAT,freshly obtained from patients, were washed in PBS threetimes by centrifugation at 300×g for 10min. After discardingPBS, 3ml of MFAT and LP was seeded in T75 flask in 9ml ofDMEM (Gibco, Life Technologies, Monza, Italy) serum-freeplain medium. The flasks were incubated for 3, 7, 14, 21,and 28 days at 37°C in 5% CO2. At the end of each incubationtime, the conditioned medium (CM) was recovered andequal amount of fresh medium was added. MFAT-CM and

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LP-CM were centrifuged at 300×g for 10min, filtered0.22μm, aliquoted, and stored at -80°C until use. MSCs from3ml MFAT or LP were isolated after collagenase (0.25% w/v,Sigma, St. Louis, MO, USA) digestion as previously described[19]. The MSCs were cultured in DMEM+10% FCS (Gibco,Dublin, Ireland) until reaching 70-80% of confluence, andthen the cells were detachedwith trypsin, counted, and seededat 1 × 106 in T75 flask in 9ml of DMEM+0.2% human serumalbumin (HAS, Baxalta InnovationsGmbH,Vienna, Austria).The MSCs-CM was prepared upon incubation at 37°C for 3days. Prolonged incubation was not performed due to MSCapoptosis under serum-free medium culture condition.MSCs-CM was centrifuged, filtered, and stored at -80°C untilused. The schematic preparation of CM from MFAT, LP,and MSCs is also reported in Figure 1.

2.3. Quantification of Protein Content of MFAT and LPSpecimens. To quantify the protein content in MFAT andLP, 1.5ml of both specimens for each donor was used. Briefly,fresh tissues, after three washes in PBS by centrifugation(300×g, 10min), were kept ice and sonicated in PBS (withoutCa and Mg) and 500μl of protease inhibitors (Sigma, Italy).The tissue were then centrifuged at 27,000×g at 4°C for20min. The supernatants were recovered and transferred ina new tube and analyzed for protein content by the Lowrymethod [20].

2.4. Quantification of Cells and DNA Content in MFAT andLP Specimens. 3ml of MFAT and LP specimens was used toevaluate cells and DNA content. After overnight collagenasedigestion, all the cells derived from MFAT and LP werewashed twice in PBS. Half of the final cell was then frozenand used for genomic DNA extraction using the QIAampDNA mini kit following the manufacturer’s instructionsand resuspended in 50μl of appropriate buffer (QIAGEN,Italy). In order to quantitate the approximate number of cellin each MFAT and LP sample, we followed two procedures:(1) each cell pellet was resuspended in PBS, filtered through40μm pore size to remove undigested aggregates, and thencentrifuged (300×g, 10min) and resuspended in trypan bluesolution and finally counted by hemocytometer and (2) DNAwas extracted by cell pellet of a given number of peripheralblood mononuclear cells (PBMNCs) obtained by density gra-dient centrifugation on Ficoll-Hypaque (Sigma, Italy) froman healthy volunteer. DNA concentration in each samplewas evaluated by absorbance at 260nm using a NanoDropmicrovolume spectrophotometer (Thermo Fisher, Italy).DNA samples derived by PBMNC pellets were used to drawa calibration curve by which an approximate number of cellin each pellet was calculated.

2.5. Characterization and Quantification of MSCs andCD31+/ECs in MFAT and LP Specimens. To quantify theMSCs (CD31-) and ECs (CD31+) from fresh and culturedMFAT and LP specimens, 3 to 5ml of fat samples was used.MFAT and LP specimens were cultured in DMEMserum-free medium and at days 0, 7, 14, 21, and 28 digestedwith collagenase to evaluate the total cells and MSC content.After collagenase digestion, the obtained cell pellets were

filtered through 40μm pore size and processed for CD31+

selection by using magnetic microbeads (Invitrogen, Italy,CELLection™ Pan Mouse IgG Kit,) as previously described[19]. CD31+ and CD31- cells were analyzed for endothelialand mesenchymal markers, respectively, by flow cytometry.Briefly, cells were resuspended in PBS at a concentration of1 × 105/100 μl and incubated with 10μl of conjugated pri-mary antibody for 30 min at 4°C in the dark. Phycoerythrin(PE) conjugate antibodies were used: anti-human CD34(BD Pharmingen™, San Jose, CA, USA; working dilution1 : 10) for CD31+ selected cells, anti-human CD90 (Millipore,Billerica, MA, USA; working dilution 1 : 10), and anti-humanCD105 and anti-human CD73 (BD Pharmingen™, workingdilution 1 : 10) for CD31 cells. Unspecific staining was deter-mined with appropriate isotype controls. At least 20,000events were acquired for each sample on a FACS AdvantageSE (BD Biosciences, San Diego, CA, USA) flow cytometer,and the acquisition analyses were performed using a Cell-Quest software (BD Biosciences). CD31 cells were also inves-tigated for mesenchymal markers by immunocytochemicalanalysis through cytoinclusion technique [21]. Briefly, cellpellets were resuspended in 40μl of Matrigel (BD Biosci-ences, Franklin Lakes, NJ, USA) and left to jellify for onehour at 37°C. The samples were then placed in plastic boxesand fixed in 10% formalin. Cells were analyzed for theexpression of CD90, CD105, and CD73 (BD Biosciences,Franklin Lakes, NJ, USA).

2.6. Analysis of Secretome of MFAT and LP Specimens.Human cytokines/chemokines were detected using multiplexbead assays based on xMAP technology (Bio-Plex HumanCytokine 27-Plex Panel; Bio-Plex Human Group II Cytokine23-Plex Panel; Bio-Rad Laboratories, Hercules, CA, USA).The CM fromMSCswas collected after 3 days of culture whilethe CM from MFAT and LP was collected at 3, 7, 14, 21, and28 days. All CMswere assayed for a total of 48 proteins: IL-1b,IL-2, IL-4, IL-6, IL-7, CXCL8, IL-10, IL-12 (p70), IL-13,IL-15, IL-17, CCL11, β-FGF, G-CSF, GM-CSF, IFN-γ,CXCL10, CCL2, CCL3, CCL4, PDGF-BB, CCL5, TNF-α,VEGF, IL-1α, IL-3, IL-12 (p40), IL-16, IL-18, CCL27, CXCL1,HGF, IFN-α2, LIF, CCL7, M-CSF, MIF, CXCL9, β-NGF,SCF, SCGF-β, CXCL12, TNF-β, and TRAIL.

2.7. Evaluation of Anti-Inflammatory Activity of MFAT andLP. The anti-inflammatory activity of MFAT-CM and LP-CM recovered at different incubation time was tested onthe U937 monocyte/macrophage cell line (ATCC, Manassas,VA, USA). These cells were routinely maintained in RPMImedia implemented with 10% FBS and expanded twice aweek. Corning Costar Transwell 5μm pore size (Celbio,Milan, Italy) supports were used to test the effect of MFAT-CM and LP-CM on U937 migration. MCP-1 chemokine(10 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) was used aspositive chemotactic factor. For each test, 2 × 105cells in200μl of DMEM+0.2% BSA were placed on the top of themembrane insert. To evaluate spontaneous migration,500μl of control DMEM+0.2% BSA medium was added tothe lower compartment of the wells. To evaluate MFAT-CM and LP-CM activities, different dilutions were added in

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the lower compartment of each well in the presence or in theabsence of MCP-1. Migration assay was carried out for 6 h at37°C in 5% CO2, and then the membrane inserts wereremoved, fixed in 10% formalin, and stained with Wright’ssolution. Cells attached to the upper surface of the filter wereremoved with a swab, and cells migrated across the mem-brane were counted by microscopically examining the lowersurface. Reported data represent the total number of cellsfound in 10 different fields for each membrane at 40x magni-fication. Each determination was done in duplicate. ELISAkits were used to quantify the production of RANTES andMCP-1 (R&D Systems, UK, Europe) by U937 cells line underbasal culture conditions, in the presence of inflammatorystimuli (LPS 1μg/ml, Sigma, Italy) combined or not with dif-ferent dilutions of MFAT-CM and LP-CM. All the data werenormalized for 106 U937 in 24h of incubation subtractingthe basal level of the same chemokines present in theMFAT-CM and in LP-CM.

3. Results

3.1. Characterization of MFAT and LP Samples. LP wasobtained from 17 human donors; half volume of each LP

specimen was processed by Lipogems® device to obtain thecorresponding MFAT; LP and MFAT of each donor werecharacterized for protein concentration, DNA content, thetotal number of cells obtained after collagenase digestion,CD31+ % cells to estimate the number of ECs, and finallythe number of MSCs by evaluating the positive cell expres-sion for CD105+, CD90+, and CD73+ [22].

All the results are summarized in Table 1. We observed asignificant variability among donors. However, the total pro-tein concentrations, DNA content, and the total number ofcells were higher in LP compared to MFAT. Vice versa, the% of cells positive for CD31, an endothelial marker, washigher in MFAT. Interestingly, the total absolute number ofMSCs contained in LP was superior than those in MFAT,but the % of MSCs in the total number of CD31 cells waslower in LP (median value 16.6%) than in MFAT (medianvalue 26.9%). Therefore, this data confirms previous reportsshowing that in MFAT, MSCs, and ECs are more concen-trated than in LP [17]. To further confirm the presence ofMSC phenotype in the selected CD31 cell population ofMFAT and LP, CD31 cells were cultured in DMEM+10%FCS for 2 weeks and then stained by immunocytochemistryfor CD105, CD90, and CD73 mesenchymal markers. A very

3 ml of MFAT

Plating in 9mlof serum free medium(DMEM)

Centrifugation at 300 xgand freezing -80C°

Recovery the CM andchange of serum free medium at3, 7, 14, 21 and 28 days

3 ml of LP

LP-CMM

FAT-CM

Change medium

Collagenase digestion (1h)

15-20 days culture inDMEM+10% FCSto reach 70/80% Ad-MSCsconfluence

Addition of serumfree DMEM/HSA Incubation

72h

MSCs-CM

LP

MFAT

MFAT

Leci

ved

smeg

opi

Recovery of CMand cells count

LP

Figure 1: Schematic procedure for the preparation of conditioned medium (CM) from MFAT (MFAT-CM), LP (LP-CM), and Ad-MSCs(Ad-MSCs-CM). Specimens of LP and its counterpart MFAT (3ml each) were washed in PBS three times and seeded in T75 flask in 9mlof plain DMEM (without exogenous proteins implementation). At each incubation time, the medium was aspirated and replaced with anequal amount of fresh DMEM. At the end of incubation, all the CMs were centrifuged, filtered (0.2μm), and frozen (-80°C) until used. Toobtain MSCs-CM from either MFAT or LP, tissues (3ml) were digested with collagenase. The cell pellets were cultured for 15-20 days inDMEM+10% FCS. When cells reached 80% of confluence, medium was substituted with an equal volume of DMEM+0.2%HSA. After72 h, the MSCs-CM was recovered and the cells were harvested and counted. The MSCs-CM was processed as described above.

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high expression of all markers (up to 90% positivity) wasfound in CD31 cell population of both MFAT and LP(Figure 2). The clonogenicity and the differentiation poten-tial of LP and MFAT cells were confirmed in a previous pub-lished paper ([23], data not shown).

3.2. Analysis of Secretome from LP and MFAT Specimens. Toanalyze the secretome derived from MFAT and LP speci-mens as well as from their isolated MSC cultures, we used aprocedure schematically reported in Figure 1. Briefly,MFAT-CM and LP-CM were obtained by seeding an equalvolume (3ml) of MFAT and LP specimens in 9ml of DMEMserum-free medium for different incubation time (from 72hto 28 days). The CM from MSCs (MSCs-CM) was analyzedonly at 72 h of incubation because prolonged time of MSC(both isolated from MFAT and LP) incubation underserum-free culture condition induced strong cell apoptosisand mortality. Table 2 reports the secretome analysis ofMSCs-CM, MFAT-CM, and LP-CM at 72h of incubation.In all the CM, a very similar and significant amount of cyto-kines and growth factors was found. However, the CM fromisolated MSCs derived fromMFAT contained higher amountof IL6 and MCP-1 cytokines as well as VEGF and SCGF-β

growth factors when compared to MFAT-CM or LP-CM.Similar results were obtained with MSCs derived from LP(data not shown). On the contrary, MFAT-CM and LP-CMsecreted higher level of β-FGF and HGF growth factors andIL8, IL16, MIG, and MIF cytokines respect to MSCs.

Interestingly, the intense secretory activity of MFAT andLP during the first 72 h of incubation was very similar interms of quality and quantity for cytokine secretion. OnlyG-SCF was significantly higher in MFAT-CM compared toLP-CM, suggesting that the procedure of microfragmenta-tion of LP to produce MFAT did not alter the releasing path-way of cells.

In order to evaluate the secretory activity of MFAT andLP during prolonged incubation time, the analysis ofMFAT-CM and LP-CM was repeated at 7, 14, and 28 days(Table 3). After 7 days of incubation, in LP-CM, we founda higher level of cytokines if compared to MFAT-CM(32,872 ± 9854 vs. 20,039 ± 4387 pg/ml). However, the analy-sis of CM at 14 days demonstrated a dramatic and rapiddecline of cytokines and growth factor secretion by LPrespect to MFAT-CM, in which the quantity of proteinsremained stable. After 28 days of culture, the differencesbetween MFAT and LP secretome were even more evident:

Table 1: Comparative analysis of MFAT and LP.

AT (1ml) Age Gender Protein (μg/ml) DNA (ng/ml)Total cells(n × 105/ml) % CD31pos % CD31neg

TotalMSCs× 103/ml

% MSCsCD31neg/ml

MFAT

29-78 13F/4M

0.11-0.67 (0.39) 34-103 (65.5) 1.8-5.6 (3.7)54-86(72.7)∗ 14-46 (27.3)

3.7-12.4(7.2)

15-26.9(26.3)∗

LP0.31-2.46(0.82)∗ 64-128 (83.5)

2.9-8.9(5.7)∗ 40-67 (50.5)

33-60(49.5)∗ 6.6-12 (9.8) 11-19.8 (16.6)

∗p < 0 05; AT: adipose tissue; n = 17 donors analyzed.

CD90 CD105 CD73

95%

97%

85%

90%

96%

98%

ba c

ed f

MFAT

LP

Figure 2: Expression of CD90, CD105, and CD73 markers in CD31 cells derived from MFAT and LP specimens. CD31 cells isolated fromMFAT and LP specimens were cultured for 14 days. At the end of incubation, the cells were recovered, cytoincluded inMatrigel, and analyzedby immuocytochemistry. The figures (10x magnification) showsMFAT (a, b, and c) and LP (d, e, and f) staining for CD90, CD105, and CD73,respectively. The % of positive cells is reported at the lower right corner of each picture.

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MFAT continues to release a significant amount of cytokines(23,057 ± 6590 pg/ml); on the contrary, the level of cytokinessecreted by LP was dramatically reduced (706 ± 154 pg/ml).

3.3. Long-Lasting Survival of MSCs in MFAT Specimens.MFAT and LP specimens were cultured in DMEM serum-free medium and at days 0, 7, 14, 21, and 28 digested withcollagenase to evaluate the total cells and MSC content(Figure 3). The total cell number and viability resultedreduced during culture in a more evident way for LP (75%reduction from day 0 to day 14; 95% from day 0 to day 28)than for MFAT LP (25% reduction from day 0 to day 14;50% from day 0 to day 28) (Figure 3(a)).

A similar trend was observed by evaluating specificallythe MSC contents in MFAT and in LP (Figure 3(b)): inMFAT, the MSC number was stable until day 14, and after

21 and 28 days of culture, the reduction was 50% and 75%,respectively; MSC number resulted significantly higher inMFAT than in LP (Figure 3(c)).

3.4. Long-Lasting Anti-Inflammatory Activity of MFAT-CM.We investigated the anti-inflammatory activity of MFAT-CM and LP-CM at early and late time of culture on themonocyte/macrophage U937 cell line, which has been usedas model to investigate inflammation [24, 25]. We analyzedthe ability of MFAT-CM and LP-CM to affect migration ofU937 under basal condition or in the presence of MCP-1, achemokine able to stimulate their motility (Figure 4) [26].We found that both MFAT-CM and LP-CM recovered after3 days of incubation, at both dilutions (12.5% and 50%), wereable to inhibit U937 migration either in the presence or in theabsence of MCP-1 stimuli (Figure 4(a)). At day 7 of incuba-tion, the inhibitory activity of LP-CM was present only athigher concentration (50%), whereas MFAT-CM blockedmigration even at lower dilution (Figure 4(b)). At days 14and 28, LP-CM lost efficacy; in contrast, MFAT-CM contin-ued to block U937 migration (Figures 4(a) and 4(d)). Theanti-inflammatory activity of MFAT-CM and LP-CM wasalso investigated by evaluating the release of inflammatorycytokines RANTES and MCP-1 by U937 (Figure 5). TheU937 cell line was cultured for 48h in the presence of LP-CM or MFAT-CM. Similarly to those observed on migrationexperiments, the addition of both LP-CM and MFAT-CM(day 3 of culture), were very effective in reducing RANTES(Figure 5(a)) and MCP-1 (Figure 5(b)) secretion and theinhibition activity persisted also for CM recovered at day 7of culture. At days 14 and 28 of culture, the inhibitory activitywas absent for LP-CM, whereas it was still present forMFAT-CM (Figures 5(a) and 5(b)). The inhibitory activityof MFAT-CM and LP-CM was maintained also in the pres-ence of LPS (Figures 5(c) and 5(d)).

4. Discussion

The use of adipose tissue has gradually developed into anexciting new way to be used in tissue regeneration: autolo-gous fat grafting and the use of optimized SVF have becomea hot topic with potential high-value clinical translation.

Commercial SVF preparation systems have previouslyhighlighted the necessity for in-depth analysis of safety pro-filing, viable cell analysis, and ultimately clinical trials inorder to understand and finely tune the action and benefit[27]. The “quality” of fractions and their MSC content mayindeed vary from patient to patient [28]; however, so far,when used in various treatment regimens, these differenceshave not been shown to significantly alter the outcome[29]. SVF or their purified MSC content has been reportedin the literature to have potential therapeutic value [30],but a full characterization of component vs. effect is stilllacking. Fat-derived SVF and even more MSC preparationsrequire a significant tissue manipulation with difficulties tomeet the complex GMP guidelines required for their clinicapplications [31].

For all these reasons, it is necessary to develop newtechnologies, GMP compliant, that minimized fat tissue

Table 2: MFAT, LP, and MSC secretome at 72 h of incubation.

AnalytesMSCs-CM(n = 3)

MFAT-CM(n = 8)

LP-CM(n = 8)

IL-1rα 32 ± 118 39 ± 23 —

IL-6 1930 ± 658^ 76 ± 47 26 ± 16

IL-8 545 ± 65 4290 ± 2431 4112 ± 1540

IL-12p70 149 ± 34 27 ± 14 17 ± 12

β-FGF 19 ± 12 808 ± 324∗ 973± 321∗

G-CSF 178 ± 665 681 ± 437∗ 23 ± 13

GM-CSF 87 ± 21 67 ± 13 65 ± 6

MCP-1 1437 ± 432^ 105 ± 67 116 ± 56

PDGF-BB — 31 ± 19 —

RANTES 78 ± 43 197 ± 36 164 ± 25

TNF-α 23 ± 2 33 ± 18 32 ± 15

VEGF 1409 ± 564^ 302 ± 67 238 ± 65

IL-2rα 17 ± 4 32 ± 7 32 ± 3

IL-3 99 ± 34 77 ± 18 54 ± 9

IL-12p40 130 ± 35 196 ± 39 155 ± 32

IL-16 57 ± 46 612 ± 142∗ 624 ± 110∗

IL-18 14 ± 2 — 39 ± 21

CTACK 30 ± 11 32 ± 24 54 ± 9

GROa 289 ± 78^ 86 ± 56 65 ± 12

HGF 111 ± 112 4145 ± 755∗ 2505 ± 451∗

LIF 36 ± 21 65± 24 —

M-CSF 31 ± 18 109± 93 43± 4MIF 133 ± 34 8358± 2675∗∗ 9234± 1121∗∗

MIG 14 ± 5 1055± 321∗∗ 1326± 289∗∗

SCF 30 ± 23 74 ± 34 42 ± 3

SCGF-β 11283 ± 4490^^ 1972 ± 903 477 ± 112

SDF-1 189 ± 27 71 ± 25 56 ± 11

TRAIL 15 ± 5 125 ± 57 111 ± 34

^p < 0 05; ^^p < 0 01 vs. LP-CM or MFAT-CM; ∗p < 0 05 vs. MSCs-CM.n = number of samples tested.

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Table3:MFA

TandLP

secretom

eat

3,7,14,and

28days

invitro.

Analytes

3days

ofcultu

re7days

ofcultu

re14

days

ofcultu

re28

days

ofcultu

reMFA

T-C

MLP

-CM

MFA

T-C

MLP

-CM

MFA

T-C

MLP

-CM

MFA

T-C

MLP

-CM

IL-1rα

55±18

53±17

51±27

151±

78121±

43—

48±23

—

IL-6

65±23

276±67

403±98

3982

±90

∗219±87

∗7±5

95±31

3±2

IL-8

3770

±1245

2860

±890

4777

±1769

8108

±1422

∗1038

±432

512±102

463±145∗

112±46

IL-12p70

——

—220±

88114±

32—

77±14

—

β-FGF

650±231

518±198

302±96

134±

67130±

54154±56

66±25

126±85

G-C

SF398±123

839±234

73±54

238±

98180±

38—

——

GM-C

SF79

±32

61±34

61±43

73±13

62±25

71±34

60±35

—

MCP-1

317±145

317±78

283±98

383±

121

351±87

∗—

249±124∗

PDGF-BB

47±239

32±16

——

——

——

RANTES

193±87

49±21

—114±

33—

——

—

VEGF

515±145

251±78

308±30

1446

±799∗

749±68

∗123±87

479±

39∗∗

63±18

IL-2rα

——

——

52±18

—47

±26

—

IL-3

82±43

66±31

62±15

81±34

89±25

—75

±27

−

IL-12p40

224±56

202±50

170±32

191±

87202±

4670

±31

201±86

∗—

IL-16

332±89

124±43

130±56

213±

111

58±57

—47

±15

—

GROa

68±22

102±42

134±87

787±

234

58±32

30±15

41±18

—

HGF

12003±4657

7315

±2354

5490

±1677

5584

±1989

13442±

3687

∗∗772±232

9839±2

341∗

∗239±97

LIF

56±16

56±17

61±23

177±

8932

±13

—17

±3

—

M-C

SF252±69

116±43

271±99

354±

143

124±

65—

60±9

—

MIF

7912

±2368

10086±2899

4565

±908

3382

±970

7915±7

68∗∗

1407

±398

6430±1

1387

∗∗127±54

MIG

925±329

1323

±675

1390

±455

4048

±1324

195±76

∗—

51±24

—

SCF

71±45

114±65

166±77

205±

5441

±32

—22

±9

—

SCGF-β

5591

±1457

1343

±630

1203

±544

2492

±545

5699±1

875∗

∗201±54

4560±7

99∗∗

—

SDF-1

72±21

82±29

71±32

96±29

106±

5162

±32

99±30

—

TRAIL

85±47

46±12

37±11

57±21

41±10

——

—

Total

33,759

±6567

26,279

±5688

20,039

±4387

32,872

±9854

∗31

,059±8

975∗

∗3,329±

1254

23,057±6

590∗

∗706±

154

∗p<005;∗

∗p<001

MFA

T-C

Mvs.L

P-C

M.

7Stem Cells International

manipulation. Recently, Tremolada and colleagues [16]developed a simple self-contained mechanical technique toproduce a microfragmented adipose-derived fraction(MFAT). This enzyme-free technology is able to convertlipoaspirate (LP) into MFAT using a device named Lipo-gems® and leads to prepare MFAT that contains SVF and asignificant number of MSCs [19] and ECs. Thanks to thisprocedure MFAT can be prepared intraoperatively in a veryshort time (15-20 min) [17].

Scope of this work was to investigate the long-termsurvival and content of MSCs and ECs as well theanti-inflammatory activity of LP and its derived MFAT,in vitro, with the aim to better understand their possible

in vivo mechanism of action particularly when transplantedin inflammatory diseases [18, 32].

Here, we show that mechanical treatment of LP to obtainMFAT resulted in a significant increase of MSC survival witha prolonged secretory activity, in vitro, under serum-freecondition, for more than 4 weeks.

Secretome analysis demonstrate that MFAT produced asignificant higher level of G-CSF, SCGF-β, and HGF com-pared with LP. G-CSF production in MFAT could be associ-ated to the activation of ECs probably due to the shearingforce produced by the device. G-CSF has previously beenshown to be a critical factor in augmenting tissue regener-ation of cartilage repair [33], in dermal and epidermal

5.9

4.2

2.2

0.020.6 0.3 0.05

28211470

1x106

3x106

7x106

5x106

Cell

s num

ber

Days in vitro

0.9

(a)

⁎

⁎⁎

⁎

0

12.5

25

50

100

7Days in vitro

MSC

s x 1

03 /ml

14 21 28

(b)

LP-MSCs

7d

7d

14d

14d

21d

21d

28d

28d

78±13

101±24

82±14

40±16

38±12 23±7

12±6

< 102

MFAT-MSCs

(c)

Figure 3: Total cell number and MSC content in LP andMFAT specimens. An identical volume (3ml) of MFAT (blue line) and LP (red line)specimens was digested with collagenase upon cultivation in DMEM plain medium for 0, 7, 14, 21, and 28 days to evaluate the total cells (a)andMSC content (b). On day 0, the total cells/ml as well the MSC content were higher in LP than inMFAT (total cells LP = 6 3 ± 4 4 × 105 vs.MFAT = 3 7 ± 1 8 × 105; MSCs LP 14 9 ± 6 3 × 103 vs. MFAT10 1 ± 5 8 × 103). After 14 days of culture, both total cells and MSCs weresignificantly reduced in LP, whereas in MFAT remained stable. (c) Pictures (20x magnification) of MSCs isolated and seeded in T25 flask,from LP and MFAT specimens. At the lower right corner of the pictures, the cell number/field is reported and represent the average ± SDof 5 different fields. Eight different donors were analyzed.

8 Stem Cells International

wound healing [34], and rotator cuff healing and repair[35]. Moreover, G-CSF stimulates the production and acti-vation of MSCs, induces increased expression of stem cellgrowth factor, HGF [36], and improved tissue recruitment

capacity and anti-inflammatory status (inhibition of IL-10and TNF-α) [37].

Further examination of viability and cell number indi-cated greater stability in MFAT in terms of total cell count

⁎⁎⁎⁎

⁎⁎ ⁎⁎

⁎⁎

⁎⁎

⁎⁎

⁎⁎

Mig

ratio

n of

U93

7/fil

ed+MCP-1 (20ng/ml)3d

CM dilutions CM dilutions

LP-CMCRL

MFAT-CM

50%

20

40

60

80

100

120

140

25% 12.5% 50% 25% 12.5%

(a)

⁎⁎

⁎⁎

⁎⁎

⁎⁎

⁎ ⁎

+MCP-1 (20ng/ml)7d

CM dilutions CM dilutions

LP-CMCRL

MFAT-CMM

igra

tion

of U

937/

filed

20

40

60

80

100

120

140

50% 25% 12.5% 50% 25% 12.5%

(b)

⁎⁎

⁎⁎

⁎ ⁎

⁎

+MCP-1 (20ng/ml)

LP-CMCRL

14d

CM dilutions CM dilutions

MFAT-CM

Mig

ratio

n of

U93

7/fil

ed

20

40

60

80

100

120

140

50% 25% 12.5% 50% 25% 12.5%

o

⁎o

o

(c)

+MCP-1 (20ng/ml)28d

CM dilutions CM dilutions

Mig

ratio

n of

U93

7/fil

ed

20

40

60

80

100

120

140

50% 25% 12.5% 50% 25% 12.5%

LP-CMCRL

MFAT-CM

⁎o

⁎⁎o

⁎⁎oo

oo

⁎⁎oo

(d)

Figure 4: MFAT-CM induces a long-lasting block of U937 migration. CMs were recovered at 3, 7, 14, and 28 days from MFAT and LPspecimens cultured in DMEM serum-free medium. Migration of U937 was evaluated by using Transwell inserts. CM was placed in thelower well at different dilutions. The cells placed in the upper well and migrated through a filter (5 μm pore size) to the lower well werecounted after 6 h. MFAT-CM inhibit the U937 migration also after long-term culture, whereas LP-CM was effective until 7 days. MCP-1(20 ng/ml) was used as positive control to stimulate U937 migration. The bars represent the average ± SD of the total number of cellsfound in 10 different fields for each membrane at 40x magnification. ∗p < 0 05 and ∗∗p < 0 01 vs. CTRL; °p < 0 05 and °°p < 0 01 vs. LP-CM.

9Stem Cells International

and MSC content compared with LP. It was interesting toobserve that in MFAT, after 28 days of culture, the initialnumber of MSCs was reduced to 70%, while the release of

cytokines decreased to 30%. This data seems to suggest that,particularly in MFAT, a significant amount of cytokines ini-tially secreted by the endothelium, MSCs, and by other cells

8.000

6.000

4.000

2.000

1.000

03d 7d 15d 30d 3d 7d 15d 30d

RAN

TES

pg/m

lx 1

06 U

937

CTRLLP-CMMFAT-CM

⁎⁎

⁎⁎

⁎⁎

⁎o ⁎oo⁎

(a)

8.000

6.000

4.000

2.000

1.000

03d 7d 15d 30d 3d 7d 15d 30d

RAN

TES

pg/m

lx 1

06 U

937

+LPS

CTRLLP-CMMFAT-CM

⁎⁎

⁎⁎

⁎⁎

⁎⁎

⁎o

⁎⁎oo

(b)

5.000

4.000

3.000

2.000

1.000

03d 7d 15d 30d 3d 7d 15d 30d

RAN

TES

pg/m

lx 1

06 U

937

CTRLLP-CMMFAT-CM

⁎⁎⁎⁎

⁎⁎ ⁎oo

⁎

(c)

5.000

4.000

3.000

2.000

1.000

03d 7d 15d 30d 3d 7d 15d 30d

RAN

TES

pg/m

lx 1

06 U

937

+LPS

CTRLLP-CMMFAT-CM

⁎⁎

⁎⁎

⁎⁎o

⁎⁎o

⁎⁎oo

⁎

(d)

Figure 5: MFAT-CM inhibits RANTES and MCP-1 secretion by U937. CMs were recovered at 3, 7, 14, and 28 days from MFAT and LPspecimens cultured in DMEM serum-free medium and added to U937cells at 1 : 2 dilution. The cells were incubated for 48 h and thenmedium was recovered to evaluate RANTES (a) and MCP-1 (b) concentration. Experiments were repeated in the presence of LPS (c, d)that stimulate secretion of both chemokines by U937. MFAT-CM resulted able to inhibit the secretion of RANTES and MCP-1 by U937even in the presence of LPS (1 μg/ml) stimuli. The bars represent the average ± SD of 5 different donors analyzed. ∗p < 0 05 and ∗∗p < 0 01vs. CTRL; °p < 0 05 and °°p < 0 01.

10 Stem Cells International

(pericytes, fibroblasts) could remain entrapped in the extra-cellular matrix and is slowly released over the incubationtime due of matrix degradation. In LP, matrix degradationcould occur more rapidly and this may explain why at 7 daysthe secretome had a significant higher level of cytokines butthis level decreases quickly over time.

This “stability” may be probably associated to the struc-tural inner characteristic of MFAT: MFAT is composed bysmall aggregates of “homogeneous” size [19, 37] that natu-rally preserved their cell content, as a “bioreactor.” In fact,MFAT appears to be resistant to environmentally poor tissueculture conditions (serum free) and is capable of long-termcytokine release [38]. When the aggregates are disintegratedin syringe-based fat processing or using enzyme-based tech-niques, it results in a rapid loss in cell content and viabilityand cytokine secretion.

In this study, we further showed that MFAT-CM wasable to inhibit U937 monocyte/macrophage migration evenin the presence of MCP-1 and this ability was retained evenafter 28 days of culture; in addition, secretion of MCP-1 andRANTES was similarly and significantly reduced over thesame period even in the presence of stimulating LPS. Incontrast, LP-CM is less effective. Moreover, previous studieshave shown that stem cells can stimulate MCP-1 andRANTES and this is led to inflammation through recruit-ment of blood leukocytes in a proinflammatory environment[39, 40]. Moreover, previous study on osteoarthritic-derivedchondrocytes showed that inflammatory cytokine produc-tion (such as RANTES and MCP-1) could be modulatedby adipose-derived stem cell contact but not by their derivedconditioned medium [41]. This suggests that the potency ofsecretome derived from MFAT samples may be superior tothose obtained by untreated LP or by purify MSCs.

It is well known that vascular injury is one of the maincauses that determines “vascular activation” and conse-quently the release of angiogenic and growth factors, as wellimmune modulators and cytokines [42]. MFAT preparationis the result of LP transformation by a mechanical procedurethat breaks up the fat tissue determining a great fragmenta-tion of blood microvessels without affecting MSC and ECviability [19, 37]. In addition, Harting and colleagues haveshown that the inflammatory stimulation of MSCs improvedthe anti-inflammatory activity of their secreted EVs [14].Actually, we do not know whether the long-lasting anti-inflammatory activity of MFAT secretome can be directlyrelated to EV content and if this process can be exacerbatedby the vascular activation of MFAT specimens. However, itis known that, upon vascular injury, endothelial cells releaseEVs and molecules that play important roles in inflamma-tion, angiogenesis, and thrombosis [43, 44]. We think thatthe potent anti-inflammatory activity of MFAT secretomeis a very complex phenomenon that could probably dependto a combination of molecules and EVs released either byMSCs or the activated endothelium. Very recently, Carelliand colleagues showed that mechanical activation of fat tis-sue improves its anti-inflammatory properties [45]; there-fore, it can be affirmed that the main differences betweenLP and MFAT here described are probably due to the dif-ferent “grade” of fat tissue activation (more specifically

endothelium activation?), clearly superior in MFAT thanin LP.

LP has been shown to have therapeutic efficacy in vivo[46], and, when supplemented with SVF, resulted in betterneovascularization and immunomodulation.

MFAT may be effective because it combines a naturalstructural scaffold organization with a very well preservedSVF and an extended MSC survival also in a hostile inflam-matory microenvironment. To date, no comparative studiesin human are available to demonstrate the major efficacy ofMFAT vs. LP for inflammatory diseases. Our data seem toindicate mechanistic insights as to the benefits of MFATpreparations when compared with standardized fat aspiratesfor clinical use.

A key challenge in regenerative medicine is tissue mini-mal manipulation, and we hope that this study may openthe door to possible optimization processes associated withfat grafting operative procedures [47].

Data Availability

All the data (microscopy images, ELISA test, cell count,FACS analysis, and so on) used to support the findingsof this study are available from the corresponding authorupon request.

Conflicts of Interest

Carlo Tremolada is a founder of Lipogems InternationalSpA. Offer Zeira is a consultant in Lipogems InternationalSpA. All other authors decline any conflict of interest.

Authors’ Contributions

Sara Nava (sara.nava@istitut-besta.it) did the preparation ofCM and inflammatory experiments and manuscript prepara-tion and revision. Valeria Sordi (sordi.valeria@hsr.it) did theanalysis of secretome by Luminex technology, statistical anal-ysis, and manuscript preparation and revision. Luisa Pas-cucci (luisa.pascucci@unipg.it) did the immunocytologicalstudy. Carlo Tremolada (carlo.tremolada@gmail.com) didthe preparation of LP and MFAT specimens. Emilio Ciusani(Emilio.Ciusani@istituto-besta.it) did the protein and DNAevaluation and FACS analysis. Offer Zeira (offer@ospedale-sanmichele.it) did the manuscript revision. Moris Cadei(moris.cadei@unibs.it) did the immunohistological studiesand inflammatory experiments. Gianni Soldati (gianni.solda-ti@gmail.com) did the fat tissue management. AugustoPessina (augusto.pessina@unimi.it) did the manuscriptrevision and critical evaluations of the experiments. MarkSlevin (m.a.slevin@mmu.ac.uk) did the manuscript revision.Eugenio Parati (eugenio.parati@istituto-besta.it) did themanuscript revision. Giulio Alessandri (giulio.alessandri@istituto-besta.it) did the study design, supervision of statis-tics and data analysis, and critical revision of the manuscript.Sara Nava and Valeria Sordi contributed equally to this work.

11Stem Cells International

Acknowledgments

This manuscript is dedicated to the memory of our belovedfriend and colleague Anna Benetti. We thank Dr. AnnaBenetti for the scientific contribution; we thank TizianaGulotta and Lucia Fontana who gave a great contributionto the execution of the immune method. All these contribu-tors work in the Section of Pathological Anatomy DMMT,University of Brescia, Brescia, Italy. This work was supportedby the Italian Ministry of Health (Ricerca Corrente to GA,IRCCS Neurological Institute C. Besta Foundation).

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